Angiogenically effective unit dose of FGF-2 and method of use

ABSTRACT

The present invention has multiple aspects. In particular, in one aspect, the present invention is directed to a unit dose composition comprising 0.2 μg/kg to 48 μg/kg of an FGF-2 of SEQ ID NO:2, or an angiogenically active fragment or mutein thereof in a pharmaceutically acceptable carrier. In another aspect, the present invention is directed to a method for treating a human patient for coronary artery disease, comprising administering into one or more coronary vessels or a peripheral vein of a human patient in need of treatment for coronary artery disease a safe and angiogenically effective dose of a recombinant FGF-2, or an angiogenically active fragment or mutein thereof. The single unit dose composition of the present invention provides an angiogenic effect in a human CAD patient that lasts six months before retreatment is required. In another aspect, the present invention is directed to a method of administration which optimizes patient&#39;s safety. In this embodiment, fluids, heparin and/or rate of infusion all play a role. In another aspect, the present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of FGF-2, alone or in combination with heparin, in a therapeutically effective carrier. The magnitude and duration of benefit were unexpected; in addition benefit with the IV route was unexpected.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation application of U.S.application Ser. No. 09/385,114, filed Aug. 27, 1999, the content ofwhich is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is directed to a unit dose composition forinducing cardiac angiogenesis in a human comprising a therapeuticallyeffective amount FGF-2 or an angiogenically active fragment or muteinthereof. The present invention is also directed to a method foradministering a single unit dose composition to a human to inducecardiac angiogenesis while minimizing systemic risk to the patient. Thepresent invention is useful because the disclosed unit dose composition,and method for its administration, provide an alternative to angioplastyor surgical intervention for the treatment of coronary artery disease(CAD) and further provide an adjunct for reducing post myocardialinfarct (MI) injury in humans.

BACKGROUND OF THE INVENTION

[0003] The fibroblast growth factors (FGF) are a family of at leasteighteen structurally related polypeptides (named FGF-1 to FGF-18) thatare characterized by a high degree of affinity for proteoglycans, suchas heparin. The various FGF molecules range in size from 15-23 kD, andexhibit a broad range of biological activities in normal and malignantconditions including nerve cell adhesion and differentiation [Schubertet al., J. Cell Biol. 104:635-643 (1987)]; wound healing [U.S. Pat. No.5,439,818 (Fiddes)]; as mitogens toward many mesodermal and ectodermalcell types, as trophic factors, as differentiation inducing orinhibiting factors [Clements, et al., Oncogene 8:1311-1316 (1993)]; andas an angiogenic factor [Harada, J. Clin. Invest., 94:623-630 (1994)].Thus, the FGF family is a family of pluripotent growth factors thatstimulate to varying extents fibroblasts, smooth muscle cells,epithelial cells and neuronal cells.

[0004] When FGF is released by normal tissues, such as in fetaldevelopment or wound healing, it is subject to temporal and spatialcontrols. However, many of the members of the FGF family are alsooncogenes. Thus, in the absence of temporal and spatial controls, theyhave the potential to stimulate tumor growth by providing angiogenesis.

[0005] Coronary artery disease is a progressive condition in humanswherein one or more coronary arteries gradually become occluded throughthe buildup of plaque (atherosclerosis). The coronary arteries ofpatients having this disease are often treated by balloon angioplasty orthe insertion of stents to prop open the partially occluded arteries.Ultimately, many of these patients are required to undergo coronaryartery bypass surgery at great expense and risk. It would be desirableto provide such patients with a medicament that would enhance coronaryblood flow so as to reduce the need to undergo bypass surgery.

[0006] An even more critical situation arises in humans when a patientsuffers a myocardial infarction, wherein one or more coronary arteriesor arterioles becomes completely occluded, such as by a clot. There isan immediate need to regain circulation to the portion of the myocardiumserved by the occluded artery or arteriole. If the lost coronarycirculation is restored within hours of the onset of the infarction,much of the damage to the myocardium that is downstream from theocclusion can be prevented. The clot-dissolving drugs, such as tissueplasminogen activator (tPA), streptokinase, and urokinase, have beenproven to be useful in this instance. However, as an adjunct to the clotdissolving drugs, it would also be desirable to also obtain collateralcirculation to the damaged or occluded myocardium by angiogenesis.

[0007] Accordingly, it is an object of the present invention to providea medicament and a mode of administration that provides human patientswith cardiac angiogenesis during coronary artery disease and/or postacute myocardial infarction. More particularly, it is a further objectof the present invention to provide a therapeutic dose of an FGF and amode of administration to humans that provide the desired property ofcardiac angiogenesis, while minimizing adverse effects.

[0008] Many of the various FGF molecules have been isolated andadministered to various animal models of myocardial ischemia withvarying and often times opposite results. According to Battler et al.,“the canine model of myocardial ischemia has been criticized because ofthe abundance of naturally occurring collateral circulation, as opposedto the porcine model, which ‘excels’ in its relative paucity of naturalcollateral circulation and its resemblance to the human coronarycirculation.” Battler et al., “Intracoronary Injection of BasicFibroblast Growth Factor Enhances Angiogenesis in Infarcted SwineMyocardium,” JACC, 22(7): 2001-6 (December 1993) at page 2002, col.1.However, Battler et al., who administered bovine bFGF (i.e., FGF-2) topigs in a myocardial infarct model, considered the varying results thatare obtained from one animal species to another, and expressly disclosesthat the divergent results “thus emphasiz[e] the caution that must beexercised in extrapolating results from different animal models.”Battler et al., at page 2005, col.1. Further, Battler points out that“the dosage and mode of administration of bFGF [i.e., bovine FGF-2] mayhave profound implications for the biologic effect achieved.” Battler,et al., at page 2005, col.1. Thus, it is a further object of thisinvention to discover a dosage and a mode of administration of afibroblast growth factor that would provide for the safe and efficacioustreatment of CAD and/or post MI injury in a human patient. Moregenerally, it is an object of the present invention to provide apharmaceutical composition and method for inducing angiogenesis in ahuman heart.

SUMMARY OF THE INVENTION

[0009] The Applicants have discovered that administering a single unitdose of about 0.2 μg/kg to about 48 μg/kg of rFGF-2 or an angiogenicallyactive fragment or mutein thereof into one or more coronary vessels (IC)or a peripheral vein (IV) of a human patient in need of coronaryangiogenesis, unexpectedly provided the human patient with a rapid andtherapeutic coronary angiogenesis that resulted in an unexpectedly largeincrease (i.e., 96 and 100 seconds of increase in the mean change frombaseline for all groups at 2 and 6 months) in the treated patient'sexercise tolerance time (ETT) that persisted for an unexpectedly longduration (i.e., 6 months as of this writing). These changes shouldresult in a decreased need for standard revascularization procedures. Bythe term “coronary angiogenesis,” as used herein, is meant the formationof new blood vessels, ranging in size from capillaries to arterioleswhich act as collaterals in coronary circulation. By way of comparison,angioplasty is considered a therapeutic success if it provides anincrease in a patient's ETT of greater than 30 seconds compared to theplacebo.

[0010] Accordingly, in one aspect, the invention is directed to a unitdose of rFGF-2 comprising a safe and therapeutically effective amount ofrFGF-2 or an angiogenically active fragment or mutein thereof.Typically, the safe and therapeutically effective amount comprises about0.2 μg/kg to about 48 μg/kg of rFGF-2 or an angiogenically activefragment or mutein thereof, based upon ideal body weight. In otherembodiments, the safe and therapeutically effective amount of the unitdose comprises 0.2 μg/kg to 2.0 μg/kg, greater than 2.0 ug/kg to lessthan 24 μg/kg, or 24 μg/kg to 48 μg/kg IC of rFGF-2 or an angiogenicallyactive fragment or mutein thereof. In another embodiment, the safe andtherapeutically effective amount of the unit dose comprises 18 μg/kg to36 μg/kg IV of rFGF-2 or an angiogenically active fragment or muteinthereof. Expressed in absolute terms, the unit dose of the presentinvention comprises 0.008 mg to 7.2 mg, more typically 0.3 mg to 3.5 mg,of FGF-2 or an angiogenically active fragment or mutein thereof. Asuitable FGF-2 is the rFGF-2 of SEQ ID NO:2 or an angiogenically activefragment or mutein thereof.

[0011] In another aspect, the present invention is directed to a methodof treating a human patient for CAD or to induce coronary angiogenesistherein. The method comprises administering into one or more coronaryvessels or a peripheral vein of a human patient in need of treatment forcoronary artery disease (or in need of angiogenesis) a safe andtherapeutically effective amount of a recombinant FGF-2 (rFGF-2) or anangiogenically active fragment or mutein thereof. Typically, a portionof the safe and therapeutically effective amount is administered to eachof two coronary vessels. The safe and therapeutically effective amountcomprises about 0.2 μg/kg to about 48 μg/kg of rFGF-2 or anangiogenically active fragment or mutein thereof in a pharmaceuticallyacceptable carrier. In other embodiments, the safe and therapeuticallyeffective amount comprises 0.2 μg/kg to 2 μg/kg, >2 μg/kg to <24 μg/kg,or 24 μg/kg to 48 μg/kg of rFGF-2 an angiogenically active fragment ormutein thereof in a pharmaceutically acceptable carrier. In absoluteterms, the amount of rFGF-2 or angiogenically active fragment or muteinthereof that is used in the above method comprises 0.008 mg to 7.2 mg,more typically 0.3 mg to 3.5 mg of rFGF-2 or an angiogenically activefragment or mutein thereof.

[0012] Because FGF-2 is a glycosoaminoglycan (e.g., heparin) bindingprotein and the presence of a glycosoaminoglycan optimizes activity andAUC (see FIGS. 3 and 4), the IC dosages of RFGF-2 of the presentinvention typically are administered from 0-30 minutes prior to theadministration of a glycosoaminoglycan, such as a heparin. The heparinis administered IC or IV, typically IV. Optionally, the heparin iscombined with the unit dose composition.

[0013] Because rFGF-2 releases nitric oxide, which is a potentvasodilator, aggressive fluid management prior to (proactively) andduring the infusion is critical to patient's safety. Administration ofIV fluids (e.g., 500-1000 mL of normal saline) to establish a wedgepressure of 12 mm Hg prior to infusion and administration of boluses ofIV fluids (e.g., 200 mL normal saline) for decreases of systolic bloodpressure (e.g., <90 mm Hg) associated with infusion optimized the safetyof administration of rFGF-2 by IC or IV infusion to human patients.

[0014] Because EDTA is a potent chelator of calcium that is required fornormal myocardial contraction and cardiac conduction, minimizing theconcentration of EDTA is critical to patient's safety. A concentrationof EDTA less than 100:g/ml in the unit dose composition optimized thesafety of administration of rFGF-2 by IC or IV infusion to humanpatients.

[0015] Because a sudden bolus of rFGF-2 is associated with profoundhypotension in animals, the rate of infusion is critical to patient'ssafety. Administration at 0.5 to 2 mL per minute, typically 1 mL perminute, optimized the safety of administration of rFGF-2 by IC or IVinfusion to human patients.

[0016] The unexpected magnitude and duration of the therapeutic benefitthat was provided to human patients in need of coronary angiogenesis bythe unit dose composition and method of administration was seen as earlyas two weeks after the single unit dose was administered, and persistedfor 6 months after the single unit dose was administered IC or IV, asdetermined by measuring art-recognized clinical endpoints such as ETT,the “Seattle Angina Questionnaire” (SAQ) and MRI of the target areas ofthe heart. In particular, when the ETT of 58 human CAD patients wasassessed by treadmill at baseline, and at 1 month, 2 months, and 6months after administration of a single unit dose of rFGF-2 by IC or IVroutes, clinical benefit was observed in some patients in all dosagegroups. See Table 1. Increases in exercise capacity appear between 1 and2 months. The mean ETT increased to greater than 60 seconds at 2 and 6months with greater benefit being seen in the higher dose group (24-48μg/kg) than in the mid (6-12 μg/kg) or low (0.33-2.0 μg/kg) dose groups.(See Table 1.) Particularly unexpected and unpredicted by animal models,were the mean increases in ETT in human patients of 93.4 and 87.5seconds that were observed at 2 and 6 months, respectively, post-dosingfor those patients administered a unit dose of rFGF-2 by IV. Evenassuming a placebo effect, the mean change from baseline for the ETTseconds still allowed an unexpectedly favorable comparison of resultswith angioplasty.

[0017] When the quality of life of 48 human CAD patients was assessed bya validated, disease specific questionnaire, the Seattle AnginaQuestionnaire (SAQ), at baseline (i.e., prior to dosing), and at 2 and 6months after a single receiving a single unit dose of rFGF-2 of thepresent invention by IC or IV routes, the mean change from baseline forthe 5 scales measured by the SAQ increased in a clinically significantmanner for all dosage ranges whether administered IC or IV (Tables 2-6).In particular, the five scales assessed by the SAQ are exertionalcapacity, angina stability, angina frequency, treatment satisfaction,and disease perception. Relative to the baseline, the mean score forexertional capacity increased by 10.9 to 20.2 at 2 months; and by 16.5to 24.1 at 6 months. For angina stability, the mean score increased by32.1 to 46.2 at 2 months; and by 16.7 to 23.2 at 6 months. For anginafrequency, the mean score increased by 20.0 to 32.9 at 2 months; and by11.4 to 36.7 at 6 months. For treatment satisfaction, the mean scoreincreased by 8.5 to 19.8 at 2 months; and by 6.3 to 19.8 at 6 months.For disease perception, the mean score increased by 20.2 to 27.8 at 2months; and by 23.8 to 34.0 at 6 months. Generally, a change of 8 pointson any scale is considered clinically significant. Thus, the observedchanges of 8.5-46.2 are clinically significant for each of the fivescales that were assessed. Even assuming a placebo effect whereby a meanchange from baseline of 14 points is considered clinically significant,the results still provide for an unexpectedly superior effect at almostall scales that were assessed.

[0018] As part of this study, MRI was also performed on 33 humanpatients diagnosed with CAD to assess the effect of administering asingle unit dose of rFGF-2 on their cardiac ejection fraction, regionalmyocardial function and perfusion (delayed arrival zone). Specifically,the patients were administered a single unit dose of 0.33 μg/kg to 48μg/kg IC or 18 μg/kg to 36 μg/kg IV of rFGF-2 of SEQ ID NO:2. When the33 human CAD patients were assessed by resting cardiac magneticresonance imaging (MRI) at baseline (i.e., prior to treatment), and 1, 2and 6 months after treatment with a single unit dose of rFGF-2 of theinvention by IC or IV routes, the patients exhibited a highlystatistically significant response to the method of treatment asobjectively measured by increased target wall thickening, target wallmotion, and target area collateral extent, and by decreased target areadelayed arrival extent. By way of summary, at 1, 2 and 6 months, thetarget wall thickening increased relative to baseline at 4.4%, 6.3% and7.7%, respectively; the target wall motion increased relative tobaseline at 2.7%, 4.4% and 6.4%, respectively; the target areacollateral extent increased relative to baseline at 8.3%, 10.9% and11.2%, respectively; and the target area delayed arrival extentdecreased relative to baseline at −10.0%, −8.3% and −10.0%,respectively.

[0019] The above data demonstrates the clinical efficacy in humans ofthe present unit dose composition of rFGF-2 or an angiogenically activefragment thereof when administered IC or IV in accordance with thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1A is a plot of the mean rFGF-2 plasma concentration versustime profiles for eight different doses of rFGF-2 (SEQ ID NO:2)administered by IC infusion in humans over a 20 minute period. The eightdoses of rFGF-2 presented in FIG. 1A are 0.33, 0.65, 2, 6, 12, 24, 36,and 48 μg/kg of lean body mass (LBM).

[0021]FIG. 1B is a plot of the mean FGF-2 plasma concentration versustime profiles for 2 different doses of rFGF-2 (SEQ ID NO:2) administeredby IV infusion in humans over a 20 minute period. The two IV doses ofrFGF-2 in FIG. 1B are 18 and 36 μg/kg. The mean concentration-timeprofile following IC administration of 36 μg/kg rFGF-2 is included forcomparison.

[0022]FIG. 2 is a plot of mean FGF-2 area under the curve (AUC) inpg*min/ml corresponding to FIGS. 1A and 1B. This plot shows the doselinearity of systemic rFGF-2 exposure following IC or IV infusion. Thesystemic exposure for the IC route is similar to that observed followingIV administration.

[0023]FIG. 3 is a plot of individual human patient FGF-2 plasmaclearance (CL) values as a function of the time of heparinadministration in “minutes prior to rFGF-2 infusion” and shows theinfluence of timing of heparin administration on rFGF-2 plasma clearance(CL).

[0024]FIG. 4 is a plot individual human patient FGF-2 dose normalizedarea under curves (AUCs) as a function of the time of heparinadministration in “minutes prior to rFGF-2 infusion” and shows theinfluence of timing of heparin administration on FGF-2 AUC.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The Applicants have discovered that a single dose of rFGF-2 or anangiogenically active fragment or mutein thereof, when administered in asafe and therapeutically effective amount into one or more coronaryvessels or into a peripheral vein of a human patient diagnosed with CADprovides the patient with a safe and therapeutically efficacioustreatment for the patient's coronary artery disease that lasts at least4 to 6 months, more typically at least six months, before a furthertreatment is needed. This duration of the effect and the magnitude ofthe improvements in ETT, SAQ and MRI were unexpected for a single unitdose of medicament.

[0026] By the phrase “therapeutically effective amount” or “safe andtherapeutically effective amount” as used herein in relation to rFGF-2is meant an amount of rFGF-2 or an angiogenically active fragment ormutein thereof that when administered in accordance with this invention,is free from major complications that cannot be medically managed, andthat provides for objective cardiac improvement in patients havingsymptoms of CAD despite optimum medical management. Thus, acutehypotension that can be managed by administration of fluids, isconsidered “safe” for the purpose of this invention. Typically, the safeand therapeutically effective amount of rFGF-2 comprises about 0.2 μg/kgto about 48 μg/kg of rFGF-2 or an angiogenically active fragment ormutein thereof. A suitable FGF-2 for use in the present invention is therFGF-2 of SEQ ID NO:2 or an angiogenically active fragment or muteinthereof.

[0027] Accordingly, the present invention has multiple aspects. In itsfirst aspect, the present invention is directed to a unit dosecomposition for inducing angiogenesis in a human patient, the unit dosecomprising a therapeutically effective (i.e., an angiogenicallyeffective) amount of rFGF-2 or an angiogenically active fragment ormutein thereof, the amount comprising about 0.2 μg/kg to about 48 μg/kgof rFGF-2 or an angiogenically active fragment or mutein thereof.

[0028] By the term “unit dose composition” as used herein is meant acomposition that when administered to a human patient in accordance withthe method of the present invention provides a typical human patient inneed of angiogenesis with an angiogenic effect of significant efficacyso as not to require retreatment for at least 4-6 months, typically 6months. The unit dose composition of the present invention is typicallyprovided in combination with one or more pharmaceutically acceptableexcipients or carriers. In other embodiments of the unit dosecomposition, a safe and therapeutically effective amount comprises about0.2 μg/kg to about 2 μg/kg, about 2 μg/kg to about 24 μg/kg, or about 24μg/kg to about 48 μg/kg of rFGF-2 or an angiogenically active fragmentor mutein thereof.

[0029] It is convenient to define the unit dose composition of thepresent invention in more absolute terms that are not dependent upon theweight of the patient to be treated. When so defined, the unit dosecomposition comprises from 0.008 mg to 7.2 mg of rFGF-2 or anangiogenically active fragment or mutein thereof. In this embodiment,the unit dose composition contains a sufficient amount of FGF-2 toaccommodate dosing any one of the majority of human CAD patients,ranging from the smallest patient (e.g., 40 kg) at the lowest dosage(about 0.2 μg/kg) through the larger patients (e.g., 150 kg) at thehighest dosage (about 48 μg/kg). More typically, the unit dose comprises0.3 mg to 3.5 mg of rFGF-2 or an angiogenically active fragment ormutein thereof. The unit dose composition is typically provided insolution or lyophilized form containing the above referenced amount ofrFGF-2 and an effective amount of one or more pharmaceuticallyacceptable buffers, stabilizers and/or other excipients as laterdescribed herein.

[0030] The active agent in the Applicants' above described unit dosecomposition is a recombinant FGF-2 or an angiogenically active fragmentor mutein thereof. Methods for making recombinant FGF-2 are well-knownin the art. The recombinant FGF-2 of SEQ ID NO:2 is made as described inU.S. Pat. No. 5,155,214, entitled “Basic Fibroblast Growth Factor,”which issued on Oct. 13, 1992, and which is expressly incorporatedherein by reference in its entirety. Moreover, all other referencescited herein, whether occurring before or after this sentence, areexpressly incorporated herein by reference in their entirety. Asdisclosed in the '214 patent, a DNA of SEQ ID NO:1, which encodes a bFGF(hereinafter “FGF-2”) of SEQ ID NO:2, is inserted into a cloning vector,such as pBR322, pMB9, Col E1, pCRI, RP4 or λ-phage, and the cloningvector is used to transform either a eukaryotic or prokaryotic cell,wherein the transformed cell expresses the FGF-2. In one embodiment, thehost cell is a yeast cell, such as Saccharomyces cerevisiae. Theresulting full length FGF-2 that is expressed has 146 amino acids inaccordance with SEQ ID NO:2. Although the FGF-2 of SEQ ID NO:2 has fourcysteines, i.e., at residue positions 25, 69, 87 and 92, there are nointernal disulfide linkages. ['214 at col. 6, lines 59-61.] However, inthe event that cross-linking occurred under oxidative conditions, itwould likely occur between the residues at positions 25 and 69.

[0031] The FGF-2 of SEQ ID NO:2, which has 146 amino acid residues,differs from naturally occurring human FGF-2 by only two amino acidresidue. In particular, the amino acids at residue positions 112 and 128of the FGF-2 of SEQ ID NO:2 are Ser and Pro, respectively, whereas inhuman FGF-2, they are Thr and Ser, respectively. In nature, bovineFGF-2, like the corresponding human FGF-2 is initially synthesized invivo as a polypeptide having 155 amino acid residues. Abraham et al.“Human Basic Fibroblast Growth Factor: Nucleotide Sequence and GenomicOrganization,” EMBO J., 5(10):2523-2528 (1986). When the FGF-2 of SEQ IDNO:2 is compared to the full length 155 residue bovine FGF-2 of Abraham,the FGF-2 of SEQ ID NO:2 lacks the first nine amino acid residues, MetAla Ala Gly Ser Ile Thr Thr Leu (SEQ ID NO:3), at the N-terminus of thecorresponding full length molecule. The recombinant FGF-2 employed inthe present compositions and method was purified to pharmaceuticalquality (98% or greater purity) using the techniques described in detailin U.S. Pat. No. 4,956,455, entitled “Bovine Fibroblast Growth Factor”which issued on Sep. 11, 1990 and which is incorporated herein byreference in its entirety. In particular, the first two steps employedin the purification of the recombinant FGF-2 of Applicants' unit dosecomposition are “conventional ion-exchange and reverse phase HPLCpurification steps as described previously.” [U.S. Pat. No. 4,956,455,citing to Bolen et al., PNAS USA 81:5364-5368 (1984).] The third step,which the '455 patent refers to as the “key purification step” ['455 atcol. 7, lines 5-6], is heparin-SEPHAROSE® affinity chromatography,wherein the strong heparin binding affinity of the FGF-2 is utilized toachieve several thousand-fold purification when eluting at approximately1.4M and 1.95M NaCl ['455 at col. 9, lines 20-25]. Polypeptidehomogeneity was confirmed by reverse-phase high pressure liquidchromatography (RP-HPLC). Buffer exchange was achieved by SEPHADEX®G-25(M) gel filtration chromatography.

[0032] In addition to the 146 residue rFGF-2 of SEQ ID NO:2, the activeagent in the unit dose of the present invention also comprises an“angiogenically active fragment” of FGF-2. By the term “angiogenicallyactive fragment” of FGF-2 is meant a fragment of FGF-2 that has about80% of the 146 residues of SEQ ID NO:2 and that retains at least 50%,preferably at least 80%, of the angiogenic activity of the FGF-2 of SEQID NO:2.

[0033] To be angiogenically active, the FGF-2 fragment should have twocell binding sites and at least one of the two heparin binding sites.The two putative cell binding sites of the analogous human FGF-2 occurat residue positions 36-39 and 77-81 thereof. See Yoshida, et al.,“Genomic Sequence of hst, a Transforming Gene Encoding a ProteinHomologous to Fibroblast Growth Factors and the int-2-Encoded Protein,”PNAS USA, 84:7305-7309 (October 1987) at FIG. 3. The two putativeheparin binding sites of hFGF-2 occur at residue positions 18-22 and107-111 thereof. See Yoshida (1987) at FIG. 3. Given the greater than98% similarity between the amino acid sequences for naturally occurringhuman FGF-2 (hFGF-2) and rFGF-2 (SEQ ID NO:2), it is expected that thetwo cell binding sites for rFGF-2 (SEQ ID NO:2) are also at residuepositions 36-39 and 77-81 thereof, and that the two heparin bindingsites are at residue positions 18-22 and 107-111 thereof. Consistentwith the above, it is well known in the art that N-terminal truncationsof the FGF-2 of SEQ ID NO:2 do not eliminate its activity in cows. Inparticular, the art discloses several naturally occurring andbiologically active fragments of the FGF-2 that have N-terminaltruncations relative to the FGF-2 of SEQ ID NO:2. An active andtruncated bFGF-2 having residues 12-146 of SEQ ID NO:2 was found inbovine liver and another active and truncated bFGF-2, having residues16-146 of SEQ ID NO:2 was found in the bovine kidney, adrenal glands andtestes. [See U.S. Pat. No. 5,155,214 at col. 6, lines 41-46, citing toUeno, et al., Biochem and Biophys Res. Comm., 138:580-588 (1986).]Likewise, other fragments of the bFGF-2 of SEQ ID NO:2 that are known tohave FGF activity are FGF-2 (24-120)-OH and FGF-2 (30-110)-NH₂. [U.S.Pat. No. 5,155,214 at col. 6, lines 48-52.] These latter fragmentsretain both of the cell binding portions of FGF-2 (SEQ ID NO:2) and oneof the heparin binding segments (residues 107-111). Accordingly, theangiogenically active fragments of FGF-2 typically encompass thoseterminally truncated fragments of FGF-2 that have at least residues thatcorrespond to residues 30-110 of FGF-2 of SEQ ID NO:2; more typically,at least residues that correspond to residues 18-146 of FGF-2 of SEQ IDNO:2.

[0034] The unit dose of the present invention also comprises an“angiogenically active . . . mutein” of the rFGF-2 of SEQ ID NO:2. Bythe term “angiogenically active . . . mutein” as used herein, is meantan isolated and purified recombinant protein or polypeptide that has 65%sequence identity (homology) to any naturally occurring FGF-2, asdetermined by the Smith-Waterman homology search algorithm (Meth. Mol.Biol. 70:173-187 (1997)) as implemented in MSPRCH program (OxfordMolecular) using an affine gap search with the following searchparameters: gap open penalty of 12, and gap extension penalty of 1, andthat retains at least 50%, preferably at least 80%, of the angiogenicactivity of the naturally occurring FGF-2 with which it has said atleast 65% sequence identity. Preferably, the angiogenically activemutein has at least 75%, more preferably at least 85%, and mostpreferably, at least 90% sequence identity to the naturally occurringFGF-2. Other well-known and routinely used homology/identity scanningalgorithm programs include Pearson and Lipman, PNAS USA, 85:2444-2448(1988); Lipman and Pearson, Science, 222:1435 (1985); Devereaux et al.,Nuc. Acids Res., 12:387-395 (1984); or the BLASTP, BLASTN or BLASTXalgorithms of Altschul, et al., Mol. Biol., 215:403-410 (1990).Computerized programs using these algorithms are also available andinclude, but are not limited to: GAP, BESTFIT, BLAST, FASTA and TFASTA,which are commercially available from the Genetics Computing Group (GCG)package, Version 8, Madison Wis., USA; and CLUSTAL in the PC/Geneprogram by Intellegenetics, Mountain View Calif. Preferably, thepercentage of sequence identity is determined by using the defaultparameters determined by the program.

[0035] The phrase “sequence identity,” as used herein, is intended torefer to the percentage of the same amino acids that are found similarlypositioned within the mutein sequence when a specified, contiguoussegment of the amino acid sequence of the mutein is aligned and comparedto the amino acid sequence of the naturally occurring FGF-2.

[0036] When considering the percentage of amino acid sequence identityin the mutein, some amino acid residue positions may differ from thereference protein as a result of conservative amino acid substitutions,which do not affect the properties of the protein or protein function.In these instances, the percentage of sequence identity may be adjustedupwards to account for the similarity in conservatively substitutedamino acids. Such adjustments are well-known in the art. See, e.g.,Meyers and Miller, “Computer Applic. Bio. Sci., 4:11-17 (1988).

[0037] To prepare an “angiogenically active mutein” of an angiogenicagent of the present invention, one uses standard techniques for sitedirected mutagenesis, as known in the art and/or as taught in Gilman, etal., Gene, 8:81 (1979) or Roberts, et al., Nature, 328:731 (1987). Usingone of the site directed mutagenesis techniques, one or more pointmutations are introduced into the cDNA sequence of SEQ ID NO: 1 tointroduce one or more amino acid substitutions or an internal deletion.Conservative amino acid substitutions are those that preserve thegeneral charge, hydrophobicity/hydrophilicity, and/or steric bulk of theamino acid being substituted. By way of example, substitutions betweenthe following groups are conservative: Gly/Ala, Val/Ile/Leu, Lys/Arg,Asn/Gln, Glu/Asp, Ser/Cys/Thr, and Phe/Trp/Tyr. Significant (up to 35%)variation from the sequence of the naturally occurring angiogenic FGF-2is permitted as long as the resulting protein or polypeptide retainsangiogenic activity within the limits specified above.

[0038] Cysteine-depleted muteins are muteins within the scope of thepresent invention. These muteins are constructed using site directedmutagenesis as described above, or according to the method described inU.S. Pat. No. 4,959,314 (“the '314 patent”), entitled “Cysteine-DepletedMuteins of Biologically Active Proteins.” The '314 patent discloses howto determine biological activity and the effect of the substitution.Cysteine substitution is particularly useful in proteins having two ormore cysteines that are not involved in disulfide formation. Suitablesubstitutions include the substitution of serine for one or both of thecysteines at residue positions 87 and 92, which are not involved indisulfide formation. Preferably, substitutions are introduced at theFGF-2 N-terminus, which is not associated with angiogenic activity.However, as discussed above, conservative substitutions are suitable forintroduction throughout the molecule.

[0039] The unit dose composition of the present invention comprises asafe and an angiogenically effective dose of rFGF-2 or an angiogenicallyactive fragment or mutein thereof, and a pharmaceutically acceptablecarrier. Typically, the safe and angiogenically effective dose of thepharmaceutical composition of the present invention is in a form and asize suitable for administration to a human patient and comprises (i)0.2 μg/kg to 48 μg/kg of rFGF-2 or an angiogenically active fragment ormutein thereof, (ii) and a pharmaceutically acceptable carrier. In otherembodiments, the safe and angiogenically effective dose comprises 0.2μg/kg to 2 μg/kg, >2 μg/kg to <24 μg/kg or 24 μg/kg to 48 μg/kg of FGF-2or an angiogenically active fragment or mutein thereof, and apharmaceutically acceptable carrier. Expressed in absolute terms for themajority of human CAD patients, the unit dose of the present inventioncomprises 0.008 mg to 7.2 mg, more typically 0.3 mg to 3.5 mg, of theFGF-2 or an angiogenically active fragment or mutein thereof.

[0040] The second recited component of the unit dose composition of thepresent invention is a “pharmaceutically acceptable carrier.” By theterm “pharmaceutically acceptable carrier” as used herein is meant anyof the carriers or diluents that are well-known in the art for thestabilization and/or administration of a proteinaceous medicament thatdoes not itself induce the production of antibodies harmful to thepatient receiving the composition, and which may be administered withoutundue toxicity. The choice of the pharmaceutically acceptable carrierand its subsequent processing enables the unit dose composition of thepresent invention to be provided in either liquid or solid form.

[0041] When the unit dose composition is in liquid form, thepharmaceutically acceptable carrier comprises a stable carrier ordiluent suitable for intravenous (“IV”) or intracoronary (“IC”)injection or infusion. Suitable carriers or diluents for injectable orinfusible solutions are nontoxic to a human recipient at the dosages andconcentrations employed, and include sterile water, sugar solutions,saline solutions, protein solutions or combinations thereof.

[0042] Typically, the pharmaceutically acceptable carrier includes abuffer and one or more stabilizers, reducing agents, anti-oxidantsand/or anti-oxidant chelating agents. The use of buffers, stabilizers,reducing agents, anti-oxidants and chelating agents in the preparationof protein based compositions, particularly pharmaceutical compositions,is well-known in the art. See, Wang et al., “Review of Excipients andpHs for Parenteral Products Used in the United States, ” J. Parent. DrugAssn., 34(6):452-462 (1980); Wang et al., “Parenteral Formulations ofProteins and Peptides: Stability and Stabilizers, ” J. Parent. Sci. andTech., 42:S4-S26 (Supplement 1988); Lachman, et al., “Antioxidants andChelating Agents as Stabilizers in Liquid Dosage Forms—Part 1,” Drug andCosmetic Industry, 102(1): 36-38, 40 and 146-148 (1968); Akers, M. J.,“Antioxidants in Pharmaceutical Products,” J. Parent. Sci. and Tech.,36(5):222-228 (1988); and Methods in Enzymology, Vol. XXV, Colowick andKaplan Eds., “Reduction of Disulfide Bonds in Proteins withDithiothreitol,” by Konigsberg, pages 185-188. Suitable buffers includeacetate, adipate, benzoate, citrate, lactate, maleate, phosphate,tartarate and the salts of various amino acids. See Wang (1980) at page455. Suitable stabilizers include carbohydrates such as threlose orglycerol. Suitable reducing agents, which maintain the reduction ofreduced cysteines, include dithiothreitol (DTT also known as Cleland'sreagent) or dithioerythritol at 0.01% to 0.1% wt/wt; acetylcysteine orcysteine at 0.1% to 0.5% (pH2-3); and thioglycerol at 0.1% to 0.5% (pH3.5 to 7.0) and glutathione. See Akers (1988) at pages 225 to 226.Suitable antioxidants include sodium bisulfite, sodium sulfite, sodiummetabisulfite, sodium thiosulfate, sodium formaldehyde sulfoxylate, andascorbic acid. See Akers (1988) at pages 225. Suitable chelating agents,which chelate trace metals to prevent the trace metal catalyzedoxidation of reduced cysteines, include citrate, tartarate,ethylenediaminetetraacetic acid (EDTA) in its disodium, tetrasodium, andcalcium disodium salts, and diethylenetriamine pentaacetic acid (DTPA).See e.g., Wang (1980) at pages 457-458 and 460-461, and Akers (1988) atpages 224-227. Suitable sugars include glycerol, trehalose, glucose,galactose and mannitol, sorbitol. A suitable protein is human serumalbumin.

[0043] In liquid form, a typical unit dose composition of the presentinvention comprises from about 0.001 mg to 8 mg, more typically 0.03 to5 mg rFGF-2 or an angiogenically active fragment or mutein thereof,dissolved a pharmaceutically acceptable carrier. A suitablepharmaceutically acceptable carrier comprises 10 mM thioglycerol, 135 mMNaCl, 10 mM sodium citrate, and 1 mM EDTA, pH 5. A suitable diluent orflushing agent for the above-described unit dose composition is any ofthe above-described carriers. Typically, the diluent is the carriersolution. rFGF-2 or an angiogenically active fragment or mutein thereofis unstable for long periods of time in liquid form. To maximizestability and shelf life of the liquid form, the unit dose compositionshould be stored frozen at −60° C. When thawed, the solution is stablefor 6 months at refrigerated conditions. A typical unit dose wouldcomprise about 1-40 ml, more typically 10-40 ml, of the above-describedcomposition having 0.008-7.2 mg of rFGF-2 or an angiogenically activefragment or mutein dissolved therein. A suitable rFGF-2 for use in theunit dose is the rFGF-2 of SEQ ID NO:2 or an angiogenically activefragment or mutein thereof.

[0044] In another embodiment, the unit dose composition is provided inlyophilized (freeze-dried) form. In this form, the unit dose of rFGF-2is capable of being stored at refrigerated temperatures forsubstantially longer than 6 months without loss of therapeuticeffectiveness. Lyophilization is accomplished by the rapid freeze drying(i.e., removing water) under reduced pressure of a plurality of vials,each containing the above described liquid form of the unit dose of therFGF-2 of the present invention therein. Lyophilizers, which perform theabove described lyophilization, are commercially available and readilyoperable by those skilled in the art. The resulting lyophilized unitdose composition, in lyophilized cake form, is formulated to containwithin the resulting lyophilized cake one or more of the buffers,stabilizers, anti-oxidants, reducing agents, salts and/or sugarsdescribed above for the corresponding liquid formulation. A lyophilizedunit dose composition containing all such other components need only bereconstituted to a known volume or concentration with sterile aqueousdiluent such as sterile water, a sterile sugar solution, or a sterilesaline solution. Alternatively, it could be reconstituted with a sterilebuffer solution as described above, but lacking a chelating agent, suchas EDTA. As a lyophilized cake, the unit dose composition is stable from6 months to two years at refrigerated temperatures. Thus, storage of theunit dose composition in lyophilized form is readily accommodated usingconventional refrigeration equipment.

[0045] Because the unit dose composition of the present invention isadministered via a cardiac catheter or other injection device, which hasdead space, it is convenient to formulate the vial containing the unitdose composition so that it contains about 10-50% more of the rFGF-2 orangiogenically active fragment or mutein thereof than is to beadministered to the patient. For example, when the unit dose of therFGF-2 to be administered is 7.2 mg, the vial is optionally formulatedto contain up to 50% extra (e.g., a total of about 10.8 mg) of rFGF-2 orangiogenically active fragment or mutein thereof. The extra solution issuitable for filling the dead space in the delivery equipment. In analternative embodiment that does not allow for dead space, thepharmaceutical composition is loaded in the cardiac catheter in front ofa pharmaceutically acceptable buffer, diluent or carrier, which is thenused to deliver the appropriate amount of the one or more dosages to theone or more sites in the myocardium that are in need of angiogenesis.

[0046] As discussed above, the pharmaceutically acceptable carrier forthe above described unit dose composition comprises a buffer and one ormore stabilizers, reducing agents, anti-oxidants and/or anti-oxidantchelating agents. It is also within the scope of the present inventionthat the unit dose composition contain an amount of a glycosoaminoglycan(also known as a “proteoglycan” or a “mucopolysaccharide”), such asheparin, that is effective to bind to the FGF-2 and to the endothelialcell receptors so as to enhance the angiogenic effectiveness of theFGF-2 or angiogenically active fragment or mutein thereof. The amount ofheparin that is administered is about 10-80 U per kg of patient weight(U/kg), typically about 40 U/kg. Expressed in absolute terms, the totalamount of heparin administered to any one patient does not exceed 5,000U. Thus, upon reconstitution, the unit dose composition of the presentinvention would not only contain an angiogenically effective amount ofrFGF-2 or an angiogenically active fragment or mutein thereof, it wouldalso contain from about 10-80 U/kg of heparin, typically about 40 U/kg.The typical volume of diluent is from about 1 to 40 ml. While largervolumes of diluent could be used, such larger volumes would typicallyresult in longer administration times. Depending upon the weight of thepatient in kg, a single dose comprising from 0.2 μg/kg to 48 μg/kg ofthe rFGF-2 or an angiogenically active fragment or mutein thereof iswithdrawn from the vial as reconstituted product for administration tothe patient. Thus, an average 70 kg man that is being dosed at 24 μg/kgwould have a sufficient volume of the reconstituted product withdrawnfrom the vial to receive an IC infusion of (70 kg×30 μg/kg) 2100 μg(i.e., 2.1 mg).

[0047] In its second aspect, the present invention is directed to amethod for treating a human patient for CAD or MI, using the abovedescribed unit dose composition. In particular, in one embodiment, thepresent invention is directed to a method for treating a human patientfor coronary artery disease, comprising administering a safe andtherapeutically effective amount of a recombinant FGF-2 or anangiogenically active fragment or mutein thereof to one or more,typically two, patent coronary vessels or a peripheral vein of a humanpatient in need of treatment for coronary artery disease. The humanpatient in need of treatment for coronary artery disease is typically ahuman patient with coronary artery disease who remains symptomatic withangina despite optional medical management. A preferred coronary vesselis a coronary artery, although grafted saphenous veins and graftedinternal mammary arteries, as provided by coronary angioplasty, are alsosuitable. Suitable peripheral veins for administering the unit dosecomposition include those peripheral veins found throughout the humanbody that are routinely used by treating physicians and nurses foradministration of fluids and medicaments. Examples of such veins includethe cephalic, the median cubital, and the basilic of the arm.

[0048] When administered as an intracoronary (IC) infusion, the unitdose of rFGF-2 or angiogenic fragment or mutein thereof is typicallyadministered within an hour, more typically over a period of about 20minutes into one or more (typically, two) patent coronary vessels. Whenadministered over a twenty minute period, the unit dose composition istypically administered at a rate of 0.5 to 2.0 ml/minute, more typicallyat about 1 ml/minute. The coronary vessels can be native vessels orgrafts, so long as they are not occluded. The volume of the unit dose ofrFGF-2 or angiogenic fragment or mutein thereof is typically 10-40 ml;more typically 20 ml. The length of time for infusion of the unit doseis not critical and can be shortened or lengthened depending on the rateand volume of infusion.

[0049] When administered as an intravenous (IV) infusion, the unit doseof rFGF-2 or angiogenic fragment or mutein thereof is administeredtypically within an hour, more typically over a 20 minute period, into aperipheral vein using a conventional IV setup. When administered over atwenty minute period, the unit dose composition is typicallyadministered at a rate of 1 ml/minute.

[0050] In the phase I clinical trial of the above described method fortreating CAD, a single unit dose composition was administered IC or IVto human patients having CAD who remained symptomatic with anginadespite optional medical management. Because the method of the presentinvention induces angiogenesis, the method of the present inventionprovides treatment of the underlying condition in CAD or MI and notmerely transitory relief from the symptoms, such as provided bynitrates. Typically, the safe and therapeutically effective amount ofthe method of the present invention comprises 0.2 μg/kg to 48 μg/kg ofrFGF-2 or an angiogenically active fragment or mutein thereof in apharmaceutically acceptable carrier. In other embodiments, the safe andtherapeutically effective amount comprises 0.2 μg/kg to 2 μg/kg, >2μg/kg to <24 μg/kg, or 24 μg/kg to 48 μg/kg of rFGF-2 or anangiogenically active fragment or mutein thereof in a pharmaceuticallyacceptable carrier. In absolute terms, the safe and therapeuticallyeffective amount is about 0.008 mg to about 7.2 mg of rFGF-2 or anangiogenically active fragment or mutein thereof; more typically, 0.3 mgto 3.5 mg of rFGF-2 or an angiogenically active fragment or muteinthereof. A suitable rFGF-2 is the rFGF-2 of SEQ ID NO:2 or anangiogenically active fragment or mutein thereof.

[0051] In another aspect, the present invention is also directed to amethod for inducing angiogenesis in a heart of a human patientcomprising, administering a single unit dose composition of arecombinant FGF-2 or an angiogenically active fragment or mutein thereofto one or more coronary vessels or to a peripheral vein in a humanpatient in need of coronary angiogenesis, said unit dose compositioncomprising from about 0.008 mg to 7.2 mg of recombinant rFGF-2 or anangiogenically active fragment or mutein thereof in a pharmaceuticallyacceptable carrier. More typically, the unit dose composition comprisesabout 0.3-3.5 mg rFGF-2 or an angiogenically active fragment or muteinthereof in a pharmaceutically acceptable carrier. As described above, asingle unit dose composition containing a therapeutically effectiveamount of an rFGF-2 or an angiogenically fragment or mutein thereof isadministered to at least one coronary vessel of a human patient in needof angiogenesis, using standard cardiac catheterization techniquesalready known and used in the art for the intracoronary administrationof medicaments, e.g., thrombolytics, streptokinase, or radio-opaque dyesor magnetic particles used to visualize the coronary arteries. By way ofexample, a coronary catheter is inserted into an artery (e.g., femoralor subclavian) of the patient in need of treatment and the catheter ispushed forward, with visualization, until it is positioned in theappropriate coronary vessel of the patient to be treated. Using standardprecautions for maintaining a clear line, the pharmaceutical compositionin solution form is administered by infusing the unit dose substantiallycontinuously over a period of 10 to 30 minutes. Although thepharmaceutical composition of the invention could be administered over alonger period of time, the Applicants perceive no benefit and apotentially increased risk of thrombosis in doing so. Typically, aportion (e.g., one half) of the unit dose is administered in a firstcoronary vessel. Then, the catheter is repositioned into a secondsecondary coronary vessel and the remainder of the unit dose isadministered with flushing of the catheter. Using the above-describedrepositioning procedure, portions of the unit dose may be administeredto a plurality of coronary vessels until the entire unit dose has beenadministered. After administration, the catheter is withdrawn usingconventional art known protocols. In the phase I clinical trialsdescribed herein, therapeutic benefit was reported by patients as earlyas two weeks following the IC rFGF-2 administration of a single unitdose. Clinically significant improvement was demonstrable by objectivecriterion (ETT and/or SAQ) as early as 30 days following IC or IVadministration of a single unit dose of the present invention, and wasmaintained for six months following dosing. In certain patients withprogressive CAD disease, it may be necessary or appropriate toadminister additional unit doses of rFGF-2 at six or 12 month intervalsafter the initial unit dose, to overcome the progression of the CADduring that interim period. In some patients with very progressive CAD,unit doses of present invention would be readministered at 4 monthintervals. In any instance, the treating physician would be able todetermine the time, if any, for readministration based upon routineassessment of the clinical symptoms of the patient.

[0052] One of the benefits of the method of the present invention iscardiac angiogenesis. Accordingly, in another aspect, the presentinvention is directed to a method for inducing angiogenesis in a heartof a human patient, comprising administering into one or more coronaryvessels (IC) or into a peripheral vein (IV) of a human patient in needof coronary angiogenesis, a single unit dose composition comprising anangiogenically effective amount of rFGF-2 or an angiogenically activefragment or mutein thereof in a pharmaceutically acceptable carrier. Inthe above method, the angiogenically effective amount comprises about0.2 μg/kg to about 48 μg/kg (or in absolute terms about 0.008 mg toabout 7.2 mg) of a recombinant FGF-2 or an angiogenically activefragment or mutein thereof. More typically, the angiogenically effectiveamount comprises about 0.3 mg to 3.5 mg of a recombinant FGF-2 or anangiogenically active fragment or mutein thereof. A suitable rFGF-2 foruse in the above-identified method is the rFGF-2 of SEQ ID NO:2 or anangiogenically active fragment thereof. In one embodiment of the abovemethod, the unit dose composition is administered IC to patent coronaryvessels or IV to a peripheral vein. In another embodiment, the unit dosecomposition is administered with heparin as described herein.

[0053] The above described method for providing coronary angiogenesis isalso beneficial in human patients that have undergone a myocardialinfarction (MI) in one or more coronary arteries. Accordingly, inanother aspect, the present invention is also directed to a method fortreating a human patient for an MI comprising, administering into one ormore coronary vessels or into a peripheral vein of said human patient, asingle unit dose composition comprising a therapeutically effectiveamount of rFGF-2 or an angiogenically active fragment or mutein thereof.In the above method, the unit dose composition typically comprises about0.2 μg/kg to about 48 μg/kg (or in absolute terms about 0.008 mg toabout 7.2 mg) of a recombinant FGF-2 or an angiogenically activefragment or mutein thereof in a pharmaceutically acceptable carrier. Asuitable rFGF-2 for use in the above-identified method is the rFGF-2 ofSEQ ID NO:2 or an angiogenically active fragment thereof.

[0054] In the event of unstable angina or acute myocardial infarction,requiring angioplasty, the same doses of rFGF-2 or angiogenic fragmentor mutein thereof that are disclosed herein would also be useful as anadjunct therapy in treating those conditions. Accordingly, in anotheraspect, the present invention is directed to an improved method fortreating a patient for unstable angina or acute myocardial infarction,requiring angioplasty, the method comprising providing angioplasty tothe patient in need of treatment; the improvement comprisingadministering into one or more coronary vessels or into a peripheralvein of said human patient, a single unit dose composition comprising atherapeutically effective amount of rFGF-2 or an angiogenically activefragment or mutein thereof. In the above method, the unit dosecomposition comprises about 0.2 μg/kg to about 48 μg/kg (or in absoluteterms about 0.008 mg to about 7.2 mg) of a recombinant FGF-2 or anangiogenically active fragment or mutein thereof in a pharmaceuticallyacceptable carrier. A suitable rFGF-2 for use in the above-identifiedmethod is the rFGF-2 of SEQ ID NO:2 or an angiogenically active fragmentthereof.

[0055] In any of the above-described methods of the present invention,the rFGF-2 or the angiogenically active fragment or mutein thereof isassociated with release of nitric oxide, a recognized smooth muscledilator, which upon administration to the patient causes a sudden dropin the patient's blood pressure. Accordingly, in the methods of thepresent invention, it is preferable to hydrate the patient with IVfluids prior to administering the unit dose of the present invention.Moreover, for safety and tolerability of the unit dose, aggressive fluidmanagement during and after rFGF-2 administration is also preferred.Finally, it is also within the scope of the above described methods toinclude the step of administering an effective amount of aglycosoaminoglycan (also known as a “proteoglycan” or a“mucopolysaccharide”), such as heparin from 0-30 minutes prior toadministering the unit dose composition of the present invention.Typically, the effective amount of glycosaminoglycan (such as heparin)that is administered is about 10-80 U/kg, more typically, about 40 U/kg.However, the total amount of heparin administered to any one patientimmediately prior to dosing generally does not exceed 5,000 U.

[0056] Because EDTA is a potent chelator of calcium which is requiredfor normal myocardial contraction and cardiac conduction, minimizing theconcentration of EDTA is critical to patient's safety. A concentrationof EDTA less than 100:g/ml optimized the safety of administration ofrFGF-2 by IC or IV infusion to human patients.

[0057] Because a sudden bolus of rFGF-2 is associated with profoundhypotension in animals, the rate of infusion is critical to patient'ssafety. Administration at 0.5 to 2 mL per minute, typically 1 mL perminute, optimized the safety of administration of rFGF-2 by IC or IVinfusion to human patients.

[0058] A Phase I clinical trial directed to treating human patients forCAD by administering a single unit dose composition of the presentinvention was conducted and is described in Examples 1-3 herein. In thattrial, sixty-six (66) human patients diagnosed with CAD, who satisfiedthe criteria of Example 2 herein, received a single unit dose of rFGF-2in accordance with the method of the present invention. Specifically,fifty-two human patients were administered a unit dose of 0.33 μg/kg to48 μg/kg of rFGF-2 by IC infusion over about a 20 minute period.Fourteen human patients were administered a unit dose of either 18 μg/kgor 36 μg/kg of rFGF-2 by IV infusion over about a 20 minute period. The66 treated patients were then assessed relative to baseline (i.e., priorto treatment with the single unit dose), and again at 1 month, 2 monthsand 6 months after treatment with the single unit dose, using three setsof art-recognized assessment criteria: 1) changes in their exercisetolerance time (ETT); 2) the Seattle Angina Questionnaire, whichprovides an assessment based upon a mixed combination of objective andsubjective criteria; and 3) the measurement of physical changes in theheart as assessed by MRI.

[0059] For ETT of the 66 patients of the Phase I clinical trial ofExamples 1-3 was measured at baseline, and at 1 month, 2 months and 6months after dosing (with a single unit dose composition of theinvention) using a Bruce treadmill protocol. Subjects were excluded fromthe analysis if the treadmill protocol was not the same as used atbaseline. Therefore, the number of subjects varied over time. Inaddition, any patients who had emergency revascularization were excludedfrom the analysis. A dose was considered effective if the mean change inETT from baseline increased by greater than 60 seconds. The results ofthe ETT assessment are provided in Table 1. TABLE 1 Exercise ToleranceTime (ETT) - Change from Baseline Change from Change from Change fromFGF-2 Baseline at Baseline at Baseline at Dose Group One Month TwoMonths Six Months 0.33 to 2.0 μg/kg IC N = 8  N = 6  N = 5 (N = 16) 45.1sec 130.0 sec* 60.8 sec (low) (−105 to 180) (19 to 240) (−45 to 210) 6.0and 12 μg/kg IC N = 2  N = 4  N = 2  (N = 8)  −24.0 sec −2.5 sec 6.5 sec(mid) (−48 to 0) (−90 to 120) (−0 to 13) 24.0 to 48.0 μg/kg IC N = 18 N= 21 N = 16 (N = 28) 51.9 sec 107.9 sec* 133.1 sec* (high) (−188 to 399)(−30 to 385) (−195 to 386) 18.0 & 36.0 μg/kg IV N = 12 N = 12 N = 12 (N= 14) 45.1 sec 93.4 sec* 87.5 sec* (−75 to 237) (0 to 285) (−60 to 285)ALL GROUPS N = 40 N = 43 N = 35 (N = 66) 45.0 sec 96.0 sec 100.0 sec

[0060] Referring to Table 1, the mean change from baseline at one monthwas less than 60 seconds for all dose groups. However, the percentage ofpatients stopping their treadmill test because of angina decreased inall groups over time. At 2 months and 6 months after dosing, the meanchanges from baseline were greater in the high dose IC and IV groups ofpatients than in the low and mid dose IC groups. The persistence ofincreased ETT at 6 months (133.1 sec and 87.5 sec) in the high dose IC(24-48:g/kg) and IV (18 & 38:g/kg) groups, respectively, was unexpected.The greatest mean increases in ETT of 107.9 and 133.1 seconds at 2 and 6months, respectively, occurred in the high dose (24-48:g/kg) IC group.The IV group exhibited significant mean increases in ETT of 93.4 secondsand 87.5 seconds, at 2 months and 6 months respectively, which was notpredicted by the rat and pig animal models used herein. Overall, thepersistence of the effect (increase in ETT) at six months and itsmagnitude for both the IC and IV groups was wholly unexpected.

[0061] The 66 human patients of the Phase I clinical trial described inExamples 1-3 herein were also evaluated using the Seattle AnginaQuestionnaire (SAQ). The SAQ is a validated, disease-specific, qualityof life instrument which assesses the following five scales: 1)“exertional capacity”=limitation of physical activity; 2) “diseaseperception”=worry about MI; 3) “treatment satisfaction”; 4) “anginafrequency”=number of episodes and sublingual nitroglycerin usage; and 5)“angina stability”=number of episodes with most strenuous physicalactivity. The possible range of scores for each of the five scales is 0to 100 with the higher scores indicating a better quality of life.Typically, a mean change of 8 points or more between the mean baselinescores (ie., before treatment) and the post-treatment scores isrecognized as being “clinically significant.” However, in the presentanalysis, a dose was considered “effective” if the mean change in scorefrom baseline increased by greater than 14 points. The reason that 14was chosen (instead of 8) was to allow for the improvement that was seenin the placebo group at 2 months in a clinical trial of another growthfactor—VEGF.

[0062] In performing the SAQ evaluation, the patients were categorizedaccording to the same dosage groups that were evaluated for ETT, i.e.,0.33-2.0 μg/kg IC (low) 6.0-12.0 μg/kg IC (mid); 24-48 μg/kg IC (high);and 18 and 36 μg/kg IV. The questionnaire was administered to subjectsin each dosage group at baseline (prior to dosing), and at 2 months and6 months after being administered a single unit dose composition ofrFGF-2 in accordance with the method of the present invention.

[0063] The first SAQ scale is “exertional capacity.” The data onexertional capacity is summarized in Table 2 herein. TABLE 2 ExertionalCapacity (EC) - Change from Baseline FGF-2 Change from Baseline Changefrom Baseline Dose Group at Two Months at Six Months 0.33 to 2.0 μg/kgIC N = 14 N = 7  (N = 16) 15.0* (−25 to 53) 23.2* (0 to 53) 6.0 and 12μg/kg IC N = 7  N = 6  (N = 8) 20.2* (−14 to 44) 24.1 (−11 to 69) 24.0to 48.0 μg/kg IC N = 26 N = 23 (N = 28) 14.6* (−33 to 75) 22.9* (−14 to75) 18.0 and 36.0 μg/kg IV N = 12 N = 14 (N = 14) 10.9 (−8 to 67) 16.5*(−19 to 63)

[0064] As reflected in Table 2, the change from baseline in mean scoreincreased at 2 and 6 months for each of the three IC dosage groups andat 6 months for all dosage groups (IC and IV). All scores at all dosagelevels increased with time in going from 2 months to 6 months with thebest increases (23.2, 24.1, 22.9 and 16.5) relative to baseline beingseen at 6 months post-dosing.

[0065] The second SAQ scale to be evaluated was “angina stability.” Thedata summarizing the angina stability is presented in Table 3 herein.TABLE 3 Angina Stability (AS) - Change from Baseline FGF-2 Change fromBaseline Change from Baseline Dose Group at Two Months at Six Months0.33 to 2.0 μg/kg IC N = 13 N = 7  (N = 16) 46.2* (0 to 100) 21.4* (0 to50) 6.0 and 12 μg/kg IC N = 7  N = 6  (N = 8)  32.1 *(0 to 50) 16.7 (−25to 50) 24.0 to 48.0 μg/kg IC N = 27 N = 24 (N = 28) 34.3* (−25 to 75)17.7* (−25 to 75) 18.0 and 36.0 μg/kg IV N = 12 N = 14 (N = 14) 39.6* (0to 100) 23.2* (0 to 75)

[0066] According to Table 3, the change in score for angina stabilityincreased relative to baseline at both 2 and 6 months for each group.The improvements in angina stability seen at 2 months after dosing(46.2, 32.1, 34.3 and 39.6) were significantly greater than those scoresseen at 6 months (21.4, 16.7, 17.7 and 23.2). However, the scores foundat both 2 months and 6 months after dosing showed that all dosages werefound to be effective (>14) in increasing angina stability. Moreover,the magnitude of the increases and their duration for 6 months wereunexpected.

[0067] The third SAQ scale to be evaluated was “angina frequency.” Thedata summarizing the angina frequency is presented in Table 4 herein.TABLE 4 Angina Frequency (AF) - Change from Baseline FGF-2 Change fromBaseline Change from Baseline Dose Group at Two Months at Six Months0.33 to 2.0 μg/kg IC N = 14 N = 7 (N = 16) 27.9* (−10 to 80) 12.9 (−40to 50) (low) 6.0 and 12 μg/kg IC N = 7  N = 6  (N = 8) 32.9* (0 to 80)36.7 (−10 to 90) (mid) 24.0 to 48.0 μg/kg IC N = 27 N = 24 (N = 28)28.9* (−40 to 80) 25.8* (−30 to 80) (high) 18.0 and 36.0 μg/kg IV N = 12N = 14 (N = 14) 20.0* (0 to 90) 11.4 (−30 to 60) ALL GROUPS N = 60 N =51 (N = 66) 27.3 21.4

[0068] According to Table 4, the mean patient scores (27.9, 32.9, 28.9and 20.0) for angina frequency increased at 2 months (relative tobaseline) by an effective amount (>14) for all dosage groups and for allmodes of administration (IC or IV). The mean patient scores continued toincrease at 6 months only for the mid dose (6.0-12.0:g/kg) group,suggesting a peak effect at 2 months post-dosing. However, for the middose (6.0-12.0:g/kg) and high dose (24.0-48.0 :g/kg) groups, the changesat 2 months and 6 months were similar, suggesting a persistent effect at6 months on angina frequency. The third SAQ scale to be evaluated was“angina frequency.” The data summarizing the angina frequency ispresented in Table 4 herein.

[0069] The fourth SAQ scale to be evaluated was “angina frequency.” Thedata summarizing the angina frequency is presented in Table 5 herein.TABLE 5 Treatment Satisfaction (TS) - Change from Baseline FGF-2 Changefrom Baseline Change from Baseline Dose Group at Two Months at SixMonths 0.33 to 2.0 μg/kg IC N = 14 N = 7 (N = 16) 8.5* (−19 to 31) 6.3(−25 to 25) (low) 6.0 and 12 μg/kg IC N = 7  N = 6  (N = 8) 17.9 (−13 to44) 19.8 (0 to 63) (mid) 24.0 to 48.0 μg/kg IC N = 27 N = 24 (N = 28)18.8* (−38 to 69) 13.0 (−75 to 63) (high) 18.0 and 36.0 μg/kg IV N = 12N = 14 (N = 14) 19.8* (−13 to 63) 13.4* (−19 to 56)

[0070] According to Table 5, the score for treatment satisfactionincreased by an effective amount at 2 months for the mid and high doseIC groups as well as the IV group. At six months post-dosing, only thescore for the mid dose group IC had a score that was greater than 14,suggesting a peak effect for treatment satisfaction at 2 months.

[0071] The fifth SAQ scale to be evaluated was “disease perception.” Thedata summarizing the disease perception is presented in Table 6 herein.According to Table 6, the scores for disease perception increased frombaseline to scores of 20.2-29.2 at 2 months and 23.8-34.0 at 6 months.These scores showed that administering a single unit dose composition inaccordance with the method of the present invention was considered to beas effective (or more effective) at 6 months as at two months. Thesescores suggest a persistence of the effectiveness of the method of thepresent invention on disease perception out to six months followingadministration of a single unit dose composition. TABLE 6 DiseasePerception (DP) - Change from Baseline Change from Baseline Change fromBaseline Dose Group at Two Months at Six Months 0.33 to 2.0 μg/kg IC N =14 N = 7 (N = 16) 29.2* (−8 to 58) 26.2* (0 to 42) (low) 6.0 and 12μg/kg IC N = 7  N = 6  (N = 8)  20.2* (−8 to 50) 30.6* (0 to 58) (mid)24.0 to 48.0 μg/kg IC N = 27 N = 24 (N = 28) 27.8* (−33 to 92) 34.0*(−33 to 100) (high) 18.0 and 36.0 μg/kg IV N = 12 N = 14 (N = 14) 22.9*(−8 to 92) 23.8* (−8 to 75)

[0072] Up to 60 of the human patients of the Phase I clinical trialdescribed in Examples 1-3 herein were also evaluated using restingmagnetic resonance imaging (MRI) scans of their heart. The resting MRIscans were performed on the patients at baseline, and at 1 month, 2months and 6 months after dosing with a single unit dose composition ofthe present invention. The doses were considered “effective” based uponstatistical significance (p<0.05). The objective criteria assessed bythe resting MRI scans are the following: (1) ejection fraction; (2)myocardial infarct extent (%); (3) normal wall thickening (4) normalwall motion (%); (5) target wall thickening (%); (6) target wall motion(%); (7) target wall area collateral extent (%); and (8) target areadelayed arrival extent (%).

[0073] Based upon the resting MRI, no change in “ejection fraction” wasobserved at one month for any one group. The mean change from baselinefor all groups (n=33) at 1 month was an increase of 2.0% (p=0.042). Attwo months, the mean change from baseline for the low dose IC group(n=13) was an increase of 8.1% (p=0.007); and for all groups (n=54), themean change from baseline was an increase of 3.8% (p=0.001). At sixmonths, the mean change from baseline for the high dose IC group (n=19)was 5.3% (p=0.023); for the IV group (n=3) was 11.1% (p=0.087); and forall groups (n=33) was 5.7% (p=0.001).

[0074] Based upon the resting MRI, there was no statisticallysignificant change in the “myocardial infarct extent” (%) for any group,or for all groups in combination at 1 month, 2 months or 6 monthspost-dosing. When the normal wall motion (%) and normal wall thickeningwere assessed, there was no statistically significant change frombaseline at 1 month, 2 months or 6 months for any one group. However,there was a statistically significant change from baseline in targetwall motion for all groups at one (n=60), two (n=54) and six (n=33)months, which was reflected as a mean increase from baseline of 2.7%(p=0.015), 4.4% (p=<0.001) and 6.4% (p<0.001), respectively. However,there was also a statistically significant change from baseline intarget wall thickening for all groups at one (n=60), two (n=54) and six(n=33) months, which was reflected as a mean increase from baseline of4.4% (p=0.015), 6.3% (p=<0.001) and 7.7% (p<0.001), respectively.

[0075] The next criteria assessed by MRI was “target area collateralextent” (%). The mean increase from baseline in target area collateralextent for all groups was highly statistically significant at one month(n=31), two months (n=27) and six months (n=16), wherein the increaseswere 8.3% (p<0.001), 10.9% (p<0.001) and 11.2% (p<0.001), respectively.The greatest collateral extent increases were observed for the low andmid IC doses, i.e., at one month (10.4% and 18.3%, respectively), twomonths (14.7% and 18.0%, respectively) and six months (16.0% and novalue for mid dose, respectively), which was wholly unexpected. At onemonth, two months and six months post-dosing, the corresponding %increases in target area collateral extent that were observed for the IChigh dose group were 6.3%, 8.0% and 9.0%, respectively.

[0076] The final criteria assessed by MRI was “target area delayedarrival extent” (%). The mean decrease from baseline in target areadelayed arrival extent for all groups was highly statisticallysignificant at 1 month (n=60), 2 months (n=54) and 6 months (n=34),wherein the decreases were −5.8% (p<0.001), −8.3% (p<0.001) and −10.0%(p<0.001), respectively. The greatest target area delayed extentdecreases were observed for the low dose IC group, which was also highlyunexpected.

[0077] Thus, providing CAD patients with a single IC or IV infusion ofrFGF-2 in accordance with the present invention provided the patientswith a statistically significant physical improvement as objectivelymeasured by MRI and other conventional criteria.

[0078] Pharmacokinetics and Metabolism

[0079] The molecular structure of FGF-2 contains a positively chargedtail that is known to bind to proteoglycan chains (heparin andheparin-like structures) on cell surfaces and on the endothelial wall ofthe vasculature. See Moscatelli, et al., “Interaction of BasicFibroblast Growth Factor with Extracellular Matrix and Receptors,” Ann.NY Acad. Sci., 638:177-181 (1981).

[0080] The kidneys and liver are the major organs for the elimination ofrFGF-2. In particular, the kidneys have a protein cutoff of about 60 kDand thus retain serum albumin (MW 60 kD). However, FGF-2 (146 residues)has a molecular weight of about 16.5 kD. Accordingly, renal excretion isto be expected. In a radiolabelled biodistribution study of commerciallyavailable bovine FGF-2 (bFGF-2), both the liver and the kidney wereshown to contain high counts of the radiolabelled bFGF-2 at 1 hour afterrv or IC injection. In a published study, wherein another recombinantiodinated form of bFGF-2 was given to rats, the liver was identified asthe major organ of elimination. Whalen et al., “The Fate ofIntravenously Administered bFGF and the Effect of Heparin,” GrowthFactors, 1:157-164 (1989). It is also known that FGF-2 binds in thegeneral circulation to α₂-macroglobulin and that this complex isinternalized by receptors on the Kupffer cells. Whalen et al. (1989) andLaMarre et al., “Cytokine Binding and Clearance Properties ofProteinase-Activated Alpha-2-Macroglobulins,” Lab. Invest., 65:3-14(1991). Labelled FGF-2 fragments were not found in the plasma, but theywere found in the urine and corresponded in size to intracellularbreakdown products.

[0081] In preclinical testing, we determined the pharmacokinetics ofrFGF-2 (SEQ ID NO:2) after intravenous (IV) and intracoronary (IC)administration in domestic Yorkshire pigs, and after IV administrationdosing in Sprague Dawley (“SD”) rats. The pig models demonstrated linearpharmacokinetics (0.65 μg/kg -20 μg/kg) IC and IV. The terminalhalf-life of the FGF-2 in the pig model was 3-4 hours. The rat modelsdemonstrated linear pharmacokinetics over the range of 30-300 μg/kg IV.The terminal half-life of the FGF-2 in the rat model was 1 hour. Bothspecies showed plasma concentration suggesting a two-compartment model.

[0082] Likewise, in humans, the FGF-2 plasma concentrations after IVand/or IC infusion followed a biexponential curve with an initial steepslope and considerable decrease over several log scales (thedistribution phase) during the first hour, followed by a more moderatedecline (the elimination phase). FIG. 1A provides a plasma concentrationversus time curve showing these phases in humans after IC administrationof rFGF-2 of SEQ ID NO:2 as a function of each of the following eightdoses: 0.33 μg/kg, 0.65 μg/kg, 2 μg/kg, 6 μg/kg, 12 μg/kg, 24 μg/kg, 36μg/kg, and 48 μg/kg of lean body mass (LBM). FIG. 1A shows the plasmadose linearity for the eight doses of rFGF-2 that were administered byIC infusion over a twenty minute period. FIG. 1A also shows a biphasicplasma level decline, i.e., a fast distribution phase during the firsthour, followed by an elimination phase with an estimated T_(1/2) of 5-7hours. The plasma concentrations of FGF-2 of SEQ ID NO:2 were determinedby a commercially available ELISA (R&D Systems, Minneapolis Minn.) thatwas marketed for analysis of human FGF-2. The ELISA assay showed 100%cross-reactivity with the rFGF-2 of SEQ ID NO:2. Other members of theFGF family, as well as many other cytokines, were not detected by thisassay. Further, heparin does not interfere with the assay.

[0083]FIG. 1B is a plot of the mean FGF-2 plasma concentration as afunction of time for 18 μg/kg and 36 μg/kg rFGF-2 administered IV, ascompared to 36 μg/kg rFGF-2 administered IC. The plasma concentrationversus time profiles in FIG. 1B for the 36 μg/kg doses by the IV and ICroutes are superimposible. However, a first-pass effect with the ICroute is not eliminated.

[0084]FIG. 2 is a plot of mean FGF-2 area under the curve (AUC) inpg*min/ml corresponding to FIGS. 1A and 1B. FIG. 2 shows the doselinearity of systemic rFGF-2 exposure (AUC) following IC or IV infusion.In particular, FIG. 2 shows that the systemic exposure to rFGF-2following administration by the IC and IV routes is substantiallysimilar.

[0085]FIG. 3 is a plot of individual human patient plasma clearance (CL)values (ml/min/kg) as a function of the time of heparin administrationin “minutes prior to rFGF-2 infusion.” FIG. 3 shows the influence oftiming of heparin administration on FGF-2 plasma clearance (CL).Although FIG. 3 shows that administering heparin up to 100 minutes priorto rFGF-2 decreases FGF-2 clearance, the preferred time foradministering heparin is from 0-30 minutes prior the rFGF-2administration, wherein the effect of the heparin on decreasing FGF-2clearance is greatest.

[0086]FIG. 4 is a plot OF individual human patient rFGF-2 dosenormalized area under curves (AUCs) as a function of the time of heparinadministration in “minutes prior to rFGF-2 infusion” and shows theinfluence of timing of heparin administration on rFGF-2 AUC. FIG. 4shows that the greatest AUC/dose was achieved when an effective amountof a glycosoaminoglycan, such as heparin, was preadministered within 30minutes or less of IC rFGF-2 infusion, more preferably within 20 minutesor less of IC or IV rFGF-2 infusion. Typically, an effective amount of aglycosoaminoglycan is 10-80 U/kg heparin.

[0087] The mean pharmacokinetic parameters for rFGF-2 in humans as afunction of dosage and mode of administration are summarized in Table 8herein. Referring to Table 8, the T½ for FGF-2 in humans was determinedto range from 2.2±3.7 hours at low dose (0.33-2.0 μg/kg) IC to 7.0±3.5hours at a dose of 18-36 μg/kg IV; given the limitations of the assay,the terminal half-life is estimated at 5-7 hours for all groups. Theclearances of FGF-2 ranged from 13.2 to 18.2 L/hour/70 kg man. Finally,the steady state volume (V_(SS)) was determined to range from 11.3±10.4L/70 kg man to 16.8±10.7 L/70 kg man. TABLE 8 Mean rFGF-2 PK Parametersin Humans FGF-2 CL V_(ss) Dose μg/kg N Route (L/hr/70 kg) t_(½)(h) (L/70kg) 0.3-2   16 IC  18.2 ± 13.4 2.2 ± 3.7 11.3 ± 10.4  6-12  8 IC 13.2 ±7.3 3.1 ± 2.5 12.1 ± 4.9  24-48 28 IC 14.7 ± 8.3 6.3 ± 1.8 16.8 ± 10.718-36 14 IV 13.9 ± 7.9 7.0 ± 3.5 16.4 ± 8.6 

[0088] Although the binding of FGF-2 to heparin-like structures isstrong (dissociation constant ˜2×10⁻⁹ M), the binding of FGF-2 to aspecific tyrosine kinase receptor is approximately two orders ofmagnitude higher (dissociation constant ˜2×10⁻¹¹ M). Moscatelli et al.,(1991). Thus, without being bound to any theory, the complexation ofrFGF-2 with a glycosoaminoglycan, such as a heparin, might increasesignal transduction and mitogenesis, and/or protect the rFGF-2 fromenzymatic degradation.

[0089] The examples, which follow, provide more details on the selectioncriterion and the Phase I clinical trial that gave rise to the datadiscussed above.

EXAMPLE 1 Unit Dose of rFGF-2 Employed in the Phase I Clinical Trial

[0090] The rFGF-2 of SEQ ID NO:2 was formulated as a unit dose andpharmaceutical composition and administered to rats, pigs and ultimatelyto humans in the Phase I clinical trial referenced herein. The variousformulations are described below.

[0091] The rFGF-2 unit dose was provided as a liquid in 3 cc type Iglass vials with a laminated gray butyl rubber stopper and red flip-offoverseal. The rFGF-2 unit dose contained 1.2 ml of 0.3 mg/ml rFGF-2 ofSEQ ID NO:2 in 10 mM sodium citrate, 10 mM monothioglycerol, 1 mMdisodium dihydrate EDTA (molecular weight 372.2), 135 mM sodiumchloride, pH 5.0. Thus, in absolute terms, each vial (and unit dose)contained 0.36 mg rFGF-2. The vials containing the unit dose in liquidform were stored at 2° to 8° C.

[0092] The rFGF diluent was supplied in 5 cc type I glass vials with alaminated gray butyl rubber stopper and red flip-off overseal. TherFGF-2 diluent contains 10 mM sodium citrate, 10 mM monothioglycerol,135 mM sodium chloride, pH 5.0. Each vial contained 5.2 ml of rFGF-2diluent solution that was stored at 2° to 8° C.

[0093] The rFGF-2 pharmaceutical composition that was infused wasprepared by diluting the rFGF-2 unit dose with the rFGF diluent suchthat the infusion volume is 10-40 ml. In order to keep the EDTAconcentration below a preset limit of 100 μg/ml, the total infusionvolume was increased to a maximum of 40 ml when proportionately higherabsolute amounts of FGF-2 were administered to patients with higher bodyweights.

EXAMPLE 2 Selection Criteria for Patients with Coronary Artery Diseasefor Treatment with rFGF-2

[0094] The following selection criteria were applied to Phase I patientswith coronary artery disease, whose activities were limited by coronaryischemia despite optimal medical management, and who were not candidatesfor approved revascularization therapies:

[0095] Inclusion criteria: Subject is eligible if:

[0096] Male or female, greater than or equal to 18 years of age

[0097] Diagnosis of coronary artery disease (CAD)

[0098] Suboptimal candidates for approved revascularization procedures,e.g., angioplasty, stents, coronary artery bypass graft (CABG) (orrefuses those interventions)

[0099] Able to exercise at least three minutes using a modified Bruceprotocol and limited by coronary ischemia

[0100] Inducible and reversible defect of at least 20% myocardium onpharmacologically stressed thallium sestamibi scan

[0101] CBC, platelets, serum chemistry within clinically acceptablerange for required cardiac catheterization

[0102] Normal INR, or if anticoagulated with Coumadin, INR<2.0

[0103] Willing and able to give written informed consent to participatein this study, including all required study procedures and follow-upvisits

[0104] Exclusion criteria: Subject is not eligible if:

[0105] Malignancy: any history of malignancy within past ten years, withthe exception of curatively treated basal cell carcinoma

[0106] Ocular conditions:proliferative retinopathy, severenon-proliferative retinopathy, retinal vein occlusion, Eales' disease,or macular edema or funduscopy by ophthalmologist: history ofintraocular surgery within six months

[0107] Renal function: creatinine clearance below normal range adjustedfor age; protein >250 mg or microalbumin >30 mg/24 h urine

[0108] Class IV heart failure (New York Heart Association)

[0109] Ejection fraction <20% by echocardiogram, thallium scan, MRI orgated pooled blood scan (MUGA)

[0110] Hemodynamically relevant arrhythmias (e.g., ventricularfibrillation, sustained ventricular tachycardia)

[0111] Severe valvular stenosis (aortic area <1.0 cm², mitral area <1.2cm²), or severe valvular insufficiency

[0112] Marked increase in angina or unstable angina within three weeks

[0113] History of myocardial infarction (MI) within three months

[0114] History of transient ischemic attack (TIA) or stroke within sixmonths

[0115] History of CABG, angioplasty or stent within six months

[0116] History of treatment with transmyocardial laserrevascularization, rFGF-2, or vascular enodothelial growth factor (VEGF)within six months

[0117] Females of child-bearing potential or nursing mothers

[0118] Any pathological fibrosis, e.g., pulmonary fibrosis, scleroderma

[0119] Known vascular malformation, e.g., AV malformation, hemangiomas

[0120] Coexistence of any disease which might interfere with assessmentof symptoms of CAD, e.g., pericarditis, costochondritis, esophagitis,systemic vasculitis, sickle cell disease

[0121] Coexistence of any disease which limits performance of modifiedBruce protocol exercise stress test, e.g., paralysis or amputation of alower extremity, severe arthritis or lower extremities, severe chronicobstructive pulmonary disease (COPD)

[0122] Participation in clinical trials of investigational agents,devices or procedures within thirty days (or scheduled within sixty daysof study drug)

[0123] Known hypersensitivity to rFGF-2 or related compounds

[0124] Any condition which makes the subject unsuitable forparticipation in this study in the opinion of the investigator, e.g.,psychosis, severe mental retardation, inability to communicate withstudy personnel, drug or alcohol abuse.

EXAMPLE 3 Phase I Clinical Study on Recombinant FGF-2 (SEQ ID NO:2)Administered to Humans

[0125] The Phase I CAD trial of this example is an open label, doseescalation study of recombinant fibroblast growth factor-2 (rFGF-2) forsafety, tolerability and pharmacokinetics. The study was conducted attwo sites: Beth Israel Deaconess Hospital (Harvard) in Boston, Mass. andEmory University Hospital in Atlanta, Ga. Enrollment is complete.Subjects were treated with a single infusion of rFGF-2 on Day 1 andfollowed for 360 days; follow-up is not yet complete in some subjects.

[0126] The subject population consists of patients with advanced CAD whoare exercise limited by coronary ischemia and are considered suboptimalcandidates for (or do not want to undergo) one of the establishedrevascularization procedures (e.g., CABG, angioplasty—with or withoutstent). The major exclusion criteria were history or suspicion ofmalignancy, uncompensated heart failure or left ventricular ejectionfraction <20%, renal insufficiency or proteinuria, and various ocularconditions (e.g., proliferative diabetic retinopathy, severenon-proliferative retinopathy).

[0127] Sixty-six subjects have received rFGF-2 of SEQ ID NO:2 in thistrial: fifty-two received the rFGF-2 as an IC infusion and fourteenreceived it as an IV infusion. Each subject was observed in the hospitalfor at least twenty-four hours, and then followed as an outpatient for360 days with follow-up visits (Days 15, 29, 57, 180 and 360). At leastfour subjects were studied at each dose; if no subject experienceddose-limiting toxicity as defined by the protocol within six days, thedose was escalated. The drug was administered as a single 20 minuteinfusion divided between two major sources of coronary blood supply(IC), using standard techniques for positioning a catheter into thepatient's coronary artery (such as already employed in angioplasty) orin a peripheral vein (IV). The doses in μg/kg of rFGF-2 administered IC(and the number of patients) were 0.33 (n=4), 0.65 (n=4), 2.0 (n=8), 6.0(n=4), 12.0 (n=4), 24 (n=8), 36 (n=10) and 48 (n=10) of rFGF-2 of SEQ IDNO:2. The doses in μg/kg of rFGF-2 administered IV (and the number ofpatients) were 18.0 (n=4) and 36.0 (n=10) or rFGF-2 of SEQ ID NO: 2.

[0128] Angina frequency and quality of life was assessed by the SeattleAngina Questionnaire (SAQ) at a baseline (before rFGF-2 administration)and at 2 months and 6 months after rFGF-2 administration. Exercisetolerance time (ETT) was assessed by the treadmill test at 1, 2, and 6months. Rest/exercise nuclear perfusion and gated sestamibi-determinedrest ejection fraction (EF), and resting magnetic resonance imaging(MRI) were assessed at baseline, and at 1 month, 2 months and 6 monthspost rFGF-2 administration. MRI measurements which were thought toobjectively measure changes in % in cardiac function and perfusionincluded: (1) ejection fraction; (2) myocardial infarct extent (%); (3)normal wall thickening (4) normal motion (%); (5) target wall thickening(%); (6) target wall motion (%); (7) target wall area collateral extent(%); and (8) target area delayed arrival extent (%).

[0129] If one of four subjects experienced dose-limiting toxicity (asdefined by the protocol), four additional subjects were studied at thatdose; if none experienced toxicity, the dose was escalated and anothergroup was studied. The maximally tolerated dose (MTD) was defined as theIC dose which was tolerated by 9/10 subjects, i.e., 36 μg/kg IC.

[0130] Careful fluid management pre-infusion was prescribed using aSwan-Ganz catheter and vital signs were monitored frequently duringdosing. Heparin was administered IV prior to the infusion of rFGF-2 inall groups. The EDTA concentration was less than 100 μg/ml in the unitdose composition. Volume of study drug administered varied with dose andsubject's weight, and ranged from 10 ml at lower doses to 40 ml athigher doses.

[0131] Preliminary Results

[0132] The results presented here are unaudited and are based on a thirdinterim analysis for sixty-six subjects with six months follow-up forall groups (1-10) and the serious adverse events (SAE) report of Jul.29, 1999 from Chiron Drug Safety. Data collection for the last visit(Day 360) and final analysis is in progress.

[0133] The starting dose of 0.33 μg/kg IC was escalated over eightsequential groups to 48 μg/kg IC, at which dose 2 of ten subjectsexperienced dose-limiting toxicity (hypotension) as defined by theprotocol. Hypotension was manageable with fluids alone in all subjects(no vasopressors or assistive devices). At 36 μg/kg IC, only 1 of 10subjects had dose-limiting toxicity which defined this dose as themaximally tolerated dose (MTD). Two additional groups were studied by IVinfusion; four subjects of half the MTD (18 μg/kg) and ten subjects atthe MTD (36 μg/kg).

[0134] Hypotension was dose-limiting in humans, as predicted by theanimal model in Yorkshire pigs. However, 36.0 μg/kg rFGF-2 IC wastolerated in humans; whereas in pigs, 20.0 μg/kg rFGF-2 IC causedprofound hypotension in one of two animals. Better tolerability inhumans was attributed to aggressive fluid management and absence ofgeneral anesthesia.

[0135] As of Jul. 29, 1999, thirty-three serious adverse events (SAEs)have occurred in 24/66 subjects, but were not dose-related. Fifteen (15)SAEs were considered at least possibly related to rFGF-2; whenever therewas a difference between relatedness assigned by the investigator andthe medical monitor, the more conservative relationship was assigned.SAE's were multiple in five subjects: 01103 (0.33 μg/kg IC), 01106 (0.65μg/kg IC), 01113 (2.0 μg/kg IC), 01137 (36.0 μg/kg IV), 02101 (0.65μg/kg IC).

[0136] The most frequent treatment-emergent adverse events (AEs) on Day1 were transient systolic hypotension and transient bradycardia. Thehypotension was dose-dependent and occurred more frequently at dosesgreater than or equal to (≧) 24 μg/kg IC; bradycardia was notdose-dependent. Other adverse events (AEs) which were deemed at leastpossibly related and appeared dose-related occurred within the firstseveral days or week post infusion and included chest pain, shortness ofbreath, insomnia, anxiety, and nausea. These events were mild tomoderate in severity, and most did not require specific intervention.

[0137] When administered IC, the drug was administered overapproximately 20 minutes as a single infusion divided between two majorsources of coronary blood supply (IC), using standard techniques forpositioning a catheter into the patient's coronary artery (such asalready employed in angioplasty). When administered IV, the drug wasadministered as an infusion over 20 minutes into a peripheral vein.

[0138] The preliminary safety results indicate that serious events werenot dose related. Thus far, of the eight IC dosage groups, there werethree deaths in the lower dosage groups, i.e., at 0.65 μg/kg (Day 23),at 2.0 μg/kg (Day 57) and at 6.0 μg/kg (Day 63), and one death in thehighest dose group, i.e., at 48.0 μg/kg (approximately 4 monthspost-dosing). Three of the deaths were cardiac; one death was due to alarge B cell lymphoma that was diagnosed three weeks after dosing in thepatient in group 4 (6.0 μg/kg) who patient died at two monthspost-dosing.

[0139] Acute myocardial infarction (MI) occurred in four patients, i.e.,one patient from each of groups 1 (0.33 μg/kg), 3 (2.0 μg/kg), 4 (6.0μg/kg) and 7 (36.0 μg/kg). Multiple MIs occurred in two patients, i.e.,one from group 1 (0.33 μg/kg) and one from group 3 (2.0 μg/kg).Emergency revascularization procedures (CABG or angioplasty with orwithout stent) were performed during follow-up in 4 patients: one eachfrom groups 1 (0.33 μg/kg), 3 (2.0 μg/kg), 4 (6.0 μg/kg), and 7 (36.0μg/kg).

[0140] Acute hypotension, seen at higher doses during or just subsequentto infusion, was managed by administration of IV fluids without need fora vasopressor. The maximally tolerated dose (MTD) in humans was definedas 36 μg/kg IC. (In contrast, in pigs, the MTD was 6.5 μg/kg IC.) Dosesof rFGF-2 up to 48 μg/kg IC were administered in human patients withaggressive fluid management, but were defined by the protocol as “nottolerated” due to acute and/or orthostatic hypotension in two out of tenpatients. The terminal half-life of the infused rFGF-2 was estimated at5 to 7 hours.

[0141] The human patients in this study that were treated with a singleIC or IV infusion of rFGF-2 of SEQ ID NO:2 exhibited a mean increase inETT of 1.5 to 2 minutes. See Table 1. This is especially significantbecause an increase in ETT of greater than (>) 30 seconds is consideredsignificant and a benchmark for evaluating alternative therapies, suchas angioplasty. The angina frequency and quality of life, as measured bySAQ, showed a significant improvement at 2 months in all five subscalesfor the 66 patients (n=66) tested. See Tables 26. In Tables 2-6, a meanchange of 14 or more was considered “clinically significant.”

[0142] When 33 human CAD patients were assessed by resting cardiacmagnetic resonance imaging (MRI) at baseline, and at 1, 2, and 6 monthsafter receiving a single unit dose composition of the present inventionby IC or IV routes, a highly statistically significant increase wasobserved in target wall thickening, target wall motion and target areacollateral extent; a highly statistically significant decrease wasobserved in target area delayed arrival extent; and no statisticallysignificant changes were observed in normal wall motion, normal wallthickening or myocardial infarct extent.

[0143] In addition to the above criterion (iLe., ETT, SAQ, MRI), atreatment is considered very successful if the angiogenic effects lastat least six months. In the present Phase I study, the unexpectedlysuperior angiogenic effects were observed to last up to 6 months in somepatients in all dosage groups. Based upon the results already obtained,it is expected that the angiogenic effects may last twelve months ormore but do last at least six months in the patients, at which time theprocedure could be repeated, if necessary.

EXAMPLE 4 Proposed Phase II Clinical Study on rFGF-2 (SEQ ID NO:2)Administered to Humans to Treat Coronary Artery Disease

[0144] The Phase II clinical trial of rFGF-2 for treating human patientsfor coronary artery disease is performed as a double blind/placebocontrolled study having four arms: placebo, 0.3 μg/kg, 3.0 μg/kg, and 30μg/kg administered once IC. This study is ongoing and results are notyet available.

EXAMPLE 5 Unit Dose and Pharmaceutical Composition of rFGF-2 for thePhase II Human Clinical Trial

[0145] The rFGF-2 of SEQ ID NO:2 was formulated as a unit dosepharmaceutical composition for administration to humans in the Phase IIclinical trial referenced herein. The various formulations are describedbelow.

[0146] The rFGF-2 unit dose was prepared as a liquid in 5 cc type Iglass vials with a laminated gray butyl rubber stopper and red flip-offoverseal. The rFGF-2 formulation contains 0.3 mg/ml rFGF-2 of SEQ IDNO:2 in 10 mM sodium citrate, 10 mM monothioglycerol, 0.3 mM disodiumdihydrate EDTA (molecular weight 372.2), 135 mM sodium chloride, pH 5.0.Each vial contained 3.7 ml of rFGF-2 drug product solution (1.11 mgrFGF-2 per vial). The resulting unit dose in liquid form is stored atless than −60° C. The above described unit dose is diluted with the“rFGF-2 placebo.” Depending on the size of the patient, the contents ofseveral of the vials may be combined to produce a unit dose of 36 μg/kgfor the Phase II study.

[0147] The rFGF-2 placebo is supplied as a clear colorless liquid in 5cc type I glass vials with a laminated gray butyl rubber stopper and redflip-off overseal. The rFGF-2 placebo is indistinguishable in appearancefrom the drug product and has the following formulation: 10 mM sodiumcitrate, 10 mM monothioglycerol, 0.3 mM disodium dihydrate EDTA(molecular weight 372.2), 135 mM sodium chloride, pH 5.0. Each vialcontains 5.2 ml of rFGF-2 placebo solution. Unlike the unit dose, therFGF-2 placebo is stored at 2° to 8° C.

[0148] The rFGF-2 pharmaceutical composition that is infused is preparedby diluting the rFGF-2 unit dose with the rFGF diluent such that theinfusion volume is 20 ml for Phase II.

[0149] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference in its entirety.

[0150] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the present invention describedherein.

1 3 1 442 DNA Bovis bovinus CDS (1)..(438) 1 cca gcc cta cca gaa gat gggggg tcc ggg gcc ttc cca cca ggg cac 48 Pro Ala Leu Pro Glu Asp Gly GlySer Gly Ala Phe Pro Pro Gly His 1 5 10 15 ttc aaa gat cca aaa cga ctatat tgt aaa aac ggg ggg ttc ttc cta 96 Phe Lys Asp Pro Lys Arg Leu TyrCys Lys Asn Gly Gly Phe Phe Leu 20 25 30 cga atc cac cca gat ggg cga gtagat ggg gta cga gaa aaa tcc gat 144 Arg Ile His Pro Asp Gly Arg Val AspGly Val Arg Glu Lys Ser Asp 35 40 45 cca cac atc aaa cta caa cta caa gccgaa gaa cga ggg gta gta tcc 192 Pro His Ile Lys Leu Gln Leu Gln Ala GluGlu Arg Gly Val Val Ser 50 55 60 atc aaa ggg gta tgt gcc aac cga tat ctagcc atg aaa gaa gat ggg 240 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu AlaMet Lys Glu Asp Gly 65 70 75 80 cga cta cta gcc tcc aaa tgt gta acc gatgaa tgt ttc ttc ttc gaa 288 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp GluCys Phe Phe Phe Glu 85 90 95 cga cta gaa tcc aac aac tat aac acc tat cgatcc cga aaa tat tcc 336 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg SerArg Lys Tyr Ser 100 105 110 tcc tgg tat gta gcc cta aaa cga acc ggg caatat aaa cta ggg cca 384 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln TyrLys Leu Gly Pro 115 120 125 aaa acc ggg cca ggg caa aaa gcc atc cta ttccta cca atg tcc gcc 432 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe LeuPro Met Ser Ala 130 135 140 aaa tcc taag 442 Lys Ser 145 2 146 PRT Bovisbovinus 2 Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro GlyHis 1 5 10 15 Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly PhePhe Leu 20 25 30 Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu LysSer Asp 35 40 45 Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly ValVal Ser 50 55 60 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys GluAsp Gly 65 70 75 80 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys PhePhe Phe Glu 85 90 95 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser ArgLys Tyr Ser 100 105 110 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln TyrLys Leu Gly Pro 115 120 125 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu PheLeu Pro Met Ser Ala 130 135 140 Lys Ser 145 3 9 PRT Bovis bovinus 3 MetAla Ala Gly Ser Ile Thr Thr Leu 1 5

What is claimed is:
 1. A unit dose composition for inducing angiogenesisin a human, comprising about 0.008 mg to about 7.2 mg of FGF-2 or anangiogenically active fragment or an angiogenically active muteinthereof in a pharmaceutically acceptable carrier.
 2. The unit dosecomposition of claim 1, comprising 0.3 mg to 3.5 mg of said FGF-2, orsaid angiogenically active fragment or said angiogenically active muteinthereof.
 3. The unit dose composition of claim 1, wherein said FGF-2 hasthe amino acid sequence of SEQ ID NO:2.
 4. The unit dose composition ofclaim 3, comprising 0.3 mg to 3.5 mg of said FGF-2 of SEQ ID NO:2 orsaid angiogenically active fragment or said angiogenically active muteinthereof in a pharmaceutically acceptable carrier.
 5. The unit dosecomposition of claim 3, comprising about 0.008 mg to about 7.2 mg ofsaid angiogenically active mutein of said FGF-2 of SEQ ID NO:2 in apharmaceutically acceptable carrier.
 6. The unit dose composition ofclaim 5, comprising 0.3 mg to 3.5 mg of said angiogenically activemutein of said FGF-2 of SEQ ID NO:2 in a pharmaceutically acceptablecarrier.
 7. The unit dose composition of claim 3, comprising about 0.008mg to about 7.2 mg of said angiogenically active fragment of said FGF-2of SEQ ID NO:2 in a pharmaceutically acceptable carrier.
 8. The unitdose composition of claim 7, comprising 0.3 mg to 3.5 mg of saidangiogenically active fragment of said FGF-2 of SEQ ID NO:2 in apharmaceutically acceptable carrier.
 9. The unit dose composition ofclaim 3, comprising about 0.008 mg to about 7.2 mg of said FGF-2 of SEQID NO:2 in a pharmaceutically acceptable carrier.
 10. A unit dosecomposition for inducing angiogenesis in a human, comprising about 0.008mg to about 7.2 mg of FGF-2 in a pharmaceutically acceptable carrier,wherein said FGF-2 has the sequence set forth in SEQ ID NO:2.
 11. Theunit dose composition of claim 11, comprising 0.3 mg to 3.5 mg of saidFGF-2 in a pharmaceutically acceptable carrier.
 12. A unit dosecomposition for inducing angiogenesis in a human, comprising about 0.008mg to about 7.2 mg of an angiogenically active fragment of FGF-2 or anangiogenically active mutein of FGF-2, wherein said FGF-2 has thesequence set forth in SEQ ID NO:2.
 13. The unit dose composition ofclaim 12, comprising 0.3 mg to 3.5 mg of said angiogenically activefragment of said FGF-2 in a pharmaceutically acceptable carrier.
 14. Theunit dose composition of claim 12, comprising 0.3 mg to 3.5 mg of saidangiogenically active mutein of said FGF02 in a pharmaceuticallyacceptable carrier.